ABSTRACT Inner ear development requires the assembly of diverse cells from multiple embryonic lineages. The epithelial, neuronal, and glial components of the inner ear are ectoderm-derived, whereas the mesenchymal components are predominantly mesoderm-derived. A major engineering challenge is to establish multi-lineage inner ear tissues in vitro, which researchers could use to study human hearing and balance-related diseases, investigate developmental biology questions, and evaluate promising therapeutics. The routine use of patient-derived inner ear explants for research is not feasible because the human inner ear is difficult to biopsy. Therefore, our long-term goal is to define the chemical and physical signals required to recapitulate formation of functional human inner ear tissue in vitro from human pluripotent stem cells (hPSCs). This project builds upon a recent technological innovation reported by our laboratory: a multi-stage 3D culture system for generating inner ear organoids that contain sensory hair cells and neurons. Despite significant progress, there are remaining questions about how faithfully inner ear organoids mimic normal embryonic development. Moreover, there are technical hurdles that may limit integration of inner ear organoids into tissue-chip drug discovery platforms. Specifically, organoid production efficiency is variable and the full range of cell types in organoids is unclear. Moreover, our ability to track the development or physiology of inner ear sensory cells in real-time is limited. Our research plan will define a next-generation inner ear organoid system. For Aim 1, we will use high- throughput single-cell analysis to generate a cell fate map of developing inner ear organoids. In Aim 2, we will generate dual-reporter hPSC lines for real-time monitoring of inner ear organoid sensorineural networks. In Aim 3, we will engineer chemically-defined inner ear organoids with improved fidelity to mammalian development. Finally, we will verify inner ear organoid production from a set of four human induced pluripotent stem cell lines to ensure the reproducibility of our results. Together, completion of this project will deepen our characterization of the human inner ear organoid model and facilitate transfer of the technology to other research laboratories. Future investigations could pursue unexplored cell signaling mechanisms, model genetic diseases, or integrate organoids into tissue-chip systems. We anticipate that our study will provide broadly applicable insights that should aid the production of organoids of other sensory systems and should provide a powerful tool for otolaryngology research.